PROJECT SUMMARY PROJECT D PRDM9 is an important regulator of recombination and transcription; it is uniquely expressed during meiosis; and its absence arrests meiotic progress and gametogenesis. Thus Prdm9 has evolved functionally to serve specific meiotic and gametogenic requirements; however, the full range and nature of these requirements is not known. In particular, the roles of PRDMS in the sequence of meiotic events, its pattern of expression, its relationship to meiotically relevant nuclear structures and domains, and how the structure of PRDMS mediates its varied enzymatic effects are all unknown. The broad, long-term goals of this project are to resolve these issues by using both cellular and genetic strategies to identify mechanisms of when (Aim 1), where (Aim 2) and how (Aim 3) PRDMS promotes recombination site activation, and successful meiosis and spermatogenesis. The results of Aim 1 will establish which steps in meiosis are subject to PRDMS control and whether PRDMS function is required continuously or only at specific meiotic or spermatogenic substages. Because preliminary evidence suggests that PRDMS occupancy in nuclei may be transient, this is important for determining when chromatin marks are set, when recombination site selection occurs, and when transcriptional control is possible. The results of Aim 2 will determine localization of the PRDMS protein and use both PrdmQ mutants and well established mouse meiosis mutant models to determine the extent to which PRDMS function is required for or dependent on proteins crucial for meiosis, including those mediating chromosome synapsis and recombination. The results of Aim 3 will determine which functions of PRDMS in meiosis and gametogenesis require either its histone methyltransferase activity or its transcription-regulating KRAB domain. Recent results suggest that non-coding RNAs are among the transcriptional targets of PRDMS. In cooperation with goals of Projects B and C, this aim will define PRDMS-specific genomic sequence targets and functional clusters of sequences epigenetically marked and/or with transcription regulated by specific PRDMS domains and their relationship to PRDMS-dependent recombination hotspots. Together, the results of this project will complement the other Projects by setting PRDMS function into the well-defined sequence of meiotic chromosome events, more specifically resolving the molecular basis for its multiple roles, and contributing to understanding how PRDMS fits into the larger network of proteins that control meiosis and gametogenesis. An important facet of PRDMS biology addressed by this project is that PRDMS is a major determinant of fertility; the aberrant recombination and failed meiosis that occur in its absence can lead to sterility or birth defects in humans.